DESCRIPTION: (Applicant's Description) The proposal describes studies designed to improve our understanding of enzymatic catalysis through investigations of the mechanism for formation of biologically important reactive intermediates in water and at enzyme active sites. The activation of carbon acids for proton transfer by enzymes and by small molecule cofactors for enzymatic reactions will be investigated in two separate studies. (1) The mechanism for stabilization of the transition state for deprotonation of alpha-carbonyl carbon acids by triosephosphate isomerase (TIM) will be probed, because the wealth of kinetic, X-ray crystallographic, and mutagenesis data has failed to produce a consensus mechanism for this transition state stabilization. Previous studies of this enzyme have largely ignored the critic role of the utilization of intrinsic substrate binding energy in transition state stabilization. We will quantify the stabilization of the transition state for deprotonation of R-gyceraldehyde 3-phosphate (GAP) by TIM that results from the specific interactions of the protein catalyst with the phosphodianion and carbonyl portions of the substrate. Next, the mechanism for utilization of this binding energy will be probed by comparing the effects of perturbation of these binding interactions by mutagenesis, on enzyme activity for deprotonation of GAP and a minimal substrate that lacks the phosphate group. The purpose of these and related experiments is to determine whether the critical closure of the "mobile loop" of TIM over the phosphate group of bound substrate occurs imply to allow optimal transition state binding of the phosphate, or whether loop closure creates an environment at the active site in which proton transfer is intrinsically more favorable than in water. (2) The activation of the alpha-protons of amino acids for proton transfer by formation of Schiff's base adducts with a pyridoxal 5'-phosphate analog and with pyruvamide will be quantified by determining rate and equilibrium constants for deprotonation of these adducts. A comparison with the rate constants for deprotonation of these adducts when bound to pyridoxal-dependent amino acid racemases will provide a measure of the enzymatic rate acceleration for proton transfer from these poorly characterized carbon acids. Advances in the understanding of enzyme mechanisms that result from model studies of nonenzymatic reactions may prove critical for drug design (enzyme inhibitors), to the understanding of metabolic pathways and diseases, and to the resolution of other health-related questions.